SUMMARY MDA5 is a conserved innate immune receptor that detects viral RNAs during infection and activates antiviral immune response. Recent studies have shown that MDA5 can be activated not only during infection, but also under various physiological conditions in the absence of infection. Such ?sterile? inflammation can cause pathogenesis of inflammatory disorders, but at the same time, can be therapeutically beneficial, for example during cancer immunotherapies. Over the last few years, my lab has defined the molecular framework for understanding how MDA5 recognizes viral dsRNA and activates downstream signaling. We discovered that MDA5 assembles into filaments upon binding to dsRNA and that the filament formation is required for efficient dsRNA binding and downstream signal activation. Despite the progress, however, there are key gaps in our understanding of how MDA5 is activated and how its activity is regulated. That is, what is the exact identity of dsRNA that stimulates MDA5 both in the virus-infected and sterile inflammatory conditions, and what are the molecular events following MDA5 filament formation leading up to antiviral signal activation. The goal of this proposal is to address these two poorly understood aspects of MDA5 function by focusing on TRIM65, a ubiquitin (Ub) E3 ligase essential for MDA5 signaling. Previous studies from us and others showed that K63-linked polyUb chains (K63-Ubn) plays an important role in MDA5-mediated antiviral signaling. TRIM65 has been speculated to be the E3 ligase responsible for the K63-Ubn conjugation of MDA5. However, whether this is in fact the case, and if so, exactly how and when TRIM65 acts on MDA5 have been unclear. In our preliminary analysis, we found that TRIM65 directly binds MDA5, and this binding is strictly dependent on MDA5 filament formation. This observation suggests that TRIM65 plays a central role as a check-point for ligand discrimination and signal activation. Furthermore, we found that TRIM65 pull-down can be used for specific isolation of MDA5 filament assembled on agonist dsRNA, away from the inactive complexes of MDA5 bound to abundant ssRNAs. This finding promises a novel method for identifying MDA5 ligands, the long-sought-after milestone in the field. Building upon these progresses, we here propose to address two central questions on MDA5 functions, i.e. signaling mechanism (Aim 1) and RNA ligand selectivity (Aim 2), from the new perspective of TRIM65. More specifically, we will determine the structural and biochemical mechanisms by which TRIM65 activates and regulates MDA5 (Aim 1) and develop a novel TRIM65 pull-down strategy to identify the RNA ligands for MDA5. We believe that the proposed work would demonstrate how an E3 ligase can directly participate in the self vs. non-self discrimination and immune signaling processes, and would provide a model for investigating other E3 ligases in immune functions and beyond. Furthermore, our research may also guide new therapeutic strategies to target MDA5 functions and its antiviral signaling pathway.